EP1124959A1 - Durch zelloberflächenmoleküle induzierte makrophagenaktivierung - Google Patents

Durch zelloberflächenmoleküle induzierte makrophagenaktivierung

Info

Publication number
EP1124959A1
EP1124959A1 EP99971035A EP99971035A EP1124959A1 EP 1124959 A1 EP1124959 A1 EP 1124959A1 EP 99971035 A EP99971035 A EP 99971035A EP 99971035 A EP99971035 A EP 99971035A EP 1124959 A1 EP1124959 A1 EP 1124959A1
Authority
EP
European Patent Office
Prior art keywords
cell surface
cell
immunostimulatory
surface polypeptide
transformed
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
EP99971035A
Other languages
English (en)
French (fr)
Other versions
EP1124959B1 (de
Inventor
Weng Tao
Shou Wong
William F. Hickey
Joseph P. Hammang
E. Edward Baetge
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Neurotech SA
Original Assignee
Neurotech SA France
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Neurotech SA France filed Critical Neurotech SA France
Publication of EP1124959A1 publication Critical patent/EP1124959A1/de
Application granted granted Critical
Publication of EP1124959B1 publication Critical patent/EP1124959B1/de
Anticipated expiration legal-status Critical
Expired - Lifetime legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A61K9/0024—Solid, semi-solid or solidifying implants, which are implanted or injected in body tissue
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/02—Nasal agents, e.g. decongestants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/06—Antipsoriatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A61P21/02—Muscle relaxants, e.g. for tetanus or cramps
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A61P21/04—Drugs for disorders of the muscular or neuromuscular system for myasthenia gravis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/06—Antianaemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/02—Non-specific cardiovascular stimulants, e.g. drugs for syncope, antihypotensives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70582—CD71
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0006—Modification of the membrane of cells, e.g. cell decoration
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5014—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing toxicity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • G01N33/505—Cells of the immune system involving T-cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • G01N33/5055—Cells of the immune system involving macrophages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0087—Galenical forms not covered by A61K9/02 - A61K9/7023
    • A61K9/0092—Hollow drug-filled fibres, tubes of the core-shell type, coated fibres, coated rods, microtubules or nanotubes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2503/00—Use of cells in diagnostics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • C12N2510/02—Cells for production

Definitions

  • This invention relates generally to the immunoglobulins, and more particularly to a cell surface molecule-induced macrophage activation that results in the rejection by the host ofthe cell expressing the cell surface molecule.
  • Cell therapy seeks to provide biologically active molecules to a patient by implanting cells that produce the biologically active molecules into the patient.
  • unencapsulated cell therapy approaches "naked" cells are implanted.
  • Treatments include immunosuppression of the patient, preclearance from the recipient serum of natural antibodies, or administration of high doses of low molecular weight haptens to inhibit natural antibody binding to transplanted tissue.
  • researchers have proposed the alteration of cells to reduce or eliminate expression of antigen or epitopes that stimulate rejection ofthe cells or tissue by natural antibodies in a recipient.
  • mitotically active cells creates a risk of tumorigenicity ofthe implanted tissue.
  • a permselective physical barrier immunoisolates the implanted tissue from the host tissue.
  • the barrier permits passage of the desired molecules between the patient and the encapsulated tissue, but protects the encapsulated cells or tissue from destruction by the immune system ofthe patient.
  • Use of xenogeneic tissue or cells in encapsulated cell therapy also acts as a "safety feature," because encapsulated cells are rejected by the patient's immune system if the capsule breaks or ruptures.
  • a patient's immune system has several components, some of which are useful for encapsulated cell therapy and some of which are undesirable. In one component, phagocytes scavenge target cells, such as the xenogeneic cells described above.
  • ADCC antibody- dependent cell-mediated cytotoxicity
  • IgG immunoglobulin G
  • Fc receptor-mediated ADCC Another component of the immune system is the activation ofthe complement system.
  • the two pathways of complement activation (the classical and the alternative pathways) are both directed at a central step in complement activation, the cleavage of C3.
  • a single terminal pathway is the formation of a membrane attack complex (MAC).
  • the classical pathway is normally activated by antigen-antibody complexes., where certain antibodies are complement fixing (capable of binding to complement to cause activation of the classical pathway).
  • Activation ofthe classical pathway can be initiated with binding of Clq, the first factor of complement cascade, to the Fc region of immunoglobulin.
  • Clq the first factor of complement cascade
  • C5b a cascade of proteolytic events results in the activation of C5 convertase, which cleaves C5 into C5b and C5a.
  • the C5b then binds C6, C7, C8 to form a C5b-8 complex. Binding of C9 molecules to C5b-8 forms C5b-9 (the)
  • MAC MAC
  • the complement system is important in host defense, but activation at inappropriate sites or to an excessive degree can cause host tissue damage. Complement is a factor in the causation or propagation of tissue injury in numerous diseases. For encapsulated cell therapy approaches in humans, therefore, (1) unencapsulated cells should be rejected immediately by the host; (2) encapsulated cells should be non-immunogenic to the host; and (3) the cell elimination process should not lead to immunological memory ofthe host. Accordingly, it would be desirable to be able to deliver a biologically active molecule to human patients using encapsulated cells or tissue that both have a human immunological "background,” but also provide the safety feature of rejection by the patient in the event of capsule rupture or failure.
  • the invention provides novel approaches for expressing naturally type I cell surface molecules (i.e., with the carboxy terminus [C-terminus] projecting toward the cytosol and the amino-terminus [N-terminus] projecting away from the cell surface) as type II molecules (i.e., with the N-terminus projecting toward the cytosol and the C-terminus projecting away from the cell surface).
  • type II molecules i.e., with the N-terminus projecting toward the cytosol and the C-terminus projecting away from the cell surface.
  • the biological function is maintained in the type II orientation.
  • (1) cells expressing such molecules can be used as therapeutic agents and (2) a screening process can evaluate the function of novel molecules that were not previously available for testing.
  • the invention also provides a novel approach to predetermining the fate of the transformed cells.
  • the invention is new aspect of gene therapy and tumor therapy. While gene therapy is a new field, conferring cytotoxic sensitivity on tumor cells has been an area of active research.
  • the strategy described here can be used to target
  • the invention provides novel immunostimulatory cell surface polypeptides, novel recombinant polynucleotides encoding immunostimulatory cell surface polypeptides, and transformed cells containing the recombinant polynucleotides.
  • a transformed cell containing a recombinant polynucleotide expresses the encoded immunostimulatory cell surface polypeptide in a host
  • the host undergoes an immune response that results in rejection ofthe transformed cell by the host.
  • the host immune response can include the activation of phagocytes, such as macrophages, but does not include complement fixation.
  • the immunostimulatory cell surface polypeptides is a chimeric polypeptide containing the human transferrin receptor membrane domain anchors a human IgG, Fc to the surface of the cell plasma membrane in a "reversed orientation," thus mimicking the configuration of IgG during opsonization.
  • the transformed cells containing the recombinant polynucleotides ofthe invention are therapeutically useful for the treatment of many disorders.
  • the invention also provides diagnostic methods for identifying novel therapeutics.
  • the invention is a method for testing phagocytes for response to an immunostimulatory cell surface polypeptide.
  • a phagocyte is contacted in vitro with a transformed cell containing a recombinant polynucleotide.
  • the recombinant polynucleotide is a promoter operably linked with a polynucleotide coding for an immunostimulatory cell surface polypeptide, and the immunostimulatory cell surface polypeptide activates phagocytes, but does not fix complement.
  • the invention is a method for identifying a compound that modulates phagocyte response to an immunostimulatory cell surface polypeptide.
  • a phagocyte is contacted in vitro with a transformed cell containing a recombinant polynucleotide. The process is then repeated by contacting a phagocyte in vitro with a test compound and the transformed cell containing a recombinant polynucleotide.
  • the phagocytic activity ofthe phagocyte in the absence of the test compound is compared with the phagocytic activity of the phagocyte in the presence of the test compound.
  • a change in the phagocytic activity indicates that the test compound modulates phagocyte response to the immunostimulatory cell surface polypeptide.
  • the invention further provides a method for stimulating phagocyte activity.
  • a transformed cell containing a recombinant polynucleotide containing a promoter operably linked with a polynucleotide coding for an immunostimulatory cell surface polypeptide is administered to a host.
  • the stimulated phagocyte is a macrophage, especially a macrophagic tumor cell.
  • the transformed cell contains a therapeutic compound, such as an anti-tumor compound.
  • the invention provides a method for modulating an immune response in a host.
  • a transformed cell containing a recombinant polynucleotide with a promoter operably linked with a polynucleotide coding for an immunostimulatory cell surface polypeptide is administered to the host.
  • the administration stimulates an immune response to the transformed cell, because the activation of phagocytes, especially macrophages, acts to regulate both T and B lymphocytes. Macrophages engulf the transformed cell and present the antigenic determinants from the transformed cell to T cells, stimulating an immune response.
  • the cell expresses, on the cell surface, a "second antigen," such that the host produces an immune response against the second antigen from the transformed cell.
  • the immunostimulatory cell surface polypeptide enhances the cellular interaction with macrophages.
  • the second antigen is presented as a target for T-cells.
  • the transformed cell expresses the second antigen from a recombinant polynucleotide.
  • the invention provides a method for ablating undesirable target cells, such as tumor cells in a patient, by the targeted delivery ofthe recombinant polynucleotides ofthe invention followed by either constitutive or inducible expression of encoded polypeptide.
  • the delivery of the immunostimulatory cell surface polypeptide of the invention into solid tumors results in the selective phagocyte-mediated ablation of the undesirable cells.
  • the invention provides a method for the treatment of autoimmune disorders in a host, by eliminating autoreactive T-cells.
  • Transformed cells containing a recombinant polynucleotide comprising a promoter operably linked with a polynucleotide coding for an immunostimulatory cell surface polypeptide are administered to a host with an autoimmune disorder.
  • the cells express a therapeutically effective amount of immunostimulatory cell surface polypeptide from the recombinant polypeptide.
  • the immunostimulatory cell surface polypeptide contacting macrophages activates the macrophages to modulate host autoreactive T-cells, thereby reducing the T-cell autoreactivity in the host.
  • Macrophages specifically modulate Thl/Th2 responses.
  • the reactiveness of T-cells differ depending on the availability of co-stimulatory factors. Therefore, T-cells can be induced to become tolerant.
  • the invention provides a composition in which a transformed cell capable of expressing an immunostimulatory cell surface polypeptide is encapsulated in an immunoisolatory capsule.
  • the transformed cells of the invention are particularly useful when encapsulated for implantation in a human patient, because cells escaping from a ruptured capsule are destroyed by the patient's immune system.
  • a host immune response will not be triggered by the transformed cells expressing an immunostimulatory cell surface polypeptide in an intact device. In case of a device failure, however, the released cells are effectively eliminated by phagocytes without complement activation or the creation of an immune memory.
  • FIGURE 1 shows the rationale ofthe invention in one embodiment.
  • FIGURE 1 A shows the natural configuration of IgG opsonization.
  • FIGURE IB shows a naturally occurring cell surface IgG anchored by type I transmembrane domain, with carboxyl terminus facing cytosol and amino terminus facing out side of cell.
  • FIGURE IC shows a reverse cell surface Fc anchored by type II transmembrane domain, with amino terminus facing cytosol and carboxyl terminus facing out side of cell, mimicking IgG opsonization.
  • FIGURE 2 shows the expression of reverse hTR-Fc ⁇ H (SEQ ID NO:4) in BHK cells, with the plasma membrane-associated hlgG determined by an ELISA.
  • FIGURE 3 shows the effect of cell surface IgG Fc, either intact IgG or recombinant reverse Fc, on superoxide production.
  • FIGURE 3 A shows the dose-response effect of anti-serum opsonized BHK cells on superoxide production by mouse macrophages.
  • FIGURE 3B shows the effect of BHK-reverse Fc clones on superoxide production by mouse macrophages. The results are presented as V ma (nmoles O 2 /10 7 cells/min), with the numbers in parenthesis representing individual clones.
  • FIGURE 4 shows the effect of anti-Fc ⁇ Rl mAb F(ab') 2 on BHK-Fc ⁇ H-induced (SEQ ID NO:4) superoxide production in U937 cells.
  • the solid bar represents superoxide assay conditions without anti-Fc ⁇ Rl mAb treatment, and the shaded bar represents assay conditions with anti- Fc ⁇ Rl mAb pretreatment. -o-
  • FIGURE 5 shows the effect of BHK-Fc ⁇ H induced (SEQ ID NO:4) in complement activation.
  • C3a-desArg enzyme immunoassay was performed following pretreatment of human serum with BHK-WT, BHK-Fc ⁇ H induced (SEQ ID NO:4) and immune complexes (IC).
  • the invention provides novel recombinant polynucleotides encoding immunostimulatory cell surface polypeptides that, when expressed by a cell, result in rejection of that cell by the host immune system.
  • the invention also provides transformed cells containing the recombinant polynucleotides and methods for using the transformed cells.
  • a chimeric polypeptide containing the human transferrin receptor membrane domain anchors a human IgG, Fc to the surface ofthe cell plasma membrane in a "reversed orientation" (SEQ ID NO:2 and SEQ ID NO:4), thus mimicking the configuration of IgG during opsonization (FIGURES 1A and IC).
  • the human IgG ] chimeric polypeptide binds the Fc receptor (here, Fc ⁇ RI) to activate phagocytes, such as macrophages, but avoids the undesirable characteristics of also activating the complement cascade ("complement fixation").
  • Fc ⁇ RI Fc receptor
  • a chronically activated complement system can kill host cells, and accumulating evidence suggests that this mechanism can cause many degenerative diseases, including inflammation and neurodegenerative diseases.
  • Immunostimulatory cell surface polypeptide is a polypeptide, expressed on a cell surface, that is capable of stimulating an immune response against the cell in a host.
  • a "biologically active molecule” is an immunostimulatory cell surface polypeptide.
  • Polypeptides appropriate for use as immunostimulatory cell surface polypeptides include the following: a. opsonins such as IgG and C3b. b. proteins with carbohydrate residues that interact with the mannose-fucose receptor of phagocytes; c. proteins capable of recognition by receptors on scavenger macrophages; d.
  • ligands for integrins located on phagocytes; e. glycoproteins, such as integrins and selectins; and f. fucosyl transferase, which generates a Gal-Gal epitope recognized by macrophages.
  • immunostimulatory cell surface polypeptides can be used in either a full-length or a truncated form, as appropriate.
  • region and domain as used to describe an immunostimulatory cell surface polypeptide includes either a full-length immunostimulatory cell surface polypeptide or a part ofthe immunostimulatory cell surface polypeptide, such as the IgG regions and domains described below.
  • Immunoglobulin G is the preferred immunostimulatory cell surface polypeptide for use in this invention.
  • An IgG protein contains (1) an Fab region (including the VH, VL and CH, domains); (2) a hinge region, and (3) an Fc region (including the CH 2 and CH 3 domains).
  • the Fab region is the region of an antibody protein which includes the antigen-binding portions.
  • the "hinge” region is a flexible area on the immunoglobulin polypeptide that contains many residues ofthe amino acid proline and is where the Fc fragment joins one ofthe two Fab fragments.
  • the Fc region is the constant region on an immunoglobulin polypeptide; is located on the immunoglobulin heavy chains; and is not involved in binding antigens.
  • the Fc region can bind to an Fc receptor on phagocytes.
  • the amino-proximal end ofthe CH 2 domain, especially amino acids 234 to 237, is important for binding ofthe Fc region to the Fc receptor.
  • Fc receptors such as Fc ⁇ RI, are integral membrane proteins located on phagocytic white blood cells, such as macrophages.
  • the hinge region is important for regulating Fc-Fc receptor interactions, providing flexibility to the polypeptide and functioning as a spacer.
  • the immunoglobulin polynucleotide used for producing a recombinant immunostimulatory cell surface polypeptide can be from any vertebrate, such as human or mouse
  • the polynucleotide encodes an immunoglobulin having a substantial number of sequences that are of the same origin as the host.
  • the immunoglobulin is of human origin.
  • the immunoglobulin polynucleotide may code for a full length polypeptide or a fragment, such as a fragment of a larger fusion protein, which includes an immunostimulatory cell surface polypeptide and a "second cell surface polypeptide.”
  • Some advantages of using an immunoglobulin fusion protein include one or more of (1) possible increased avidity for multivalent ligands, (2) longer serum half-life, (3) the ability to activate effector cells by the Fc domain, and (4) ease of purification (for example, by protein A chromatography).
  • EXAMPLE 1 shows the construction and use of two immunoglobulin fusion proteins.
  • IgG,-Fc is expressed on the cell surface in a "reverse orientation" (see, FIGURE 2).
  • the Fc is in a reverse orientation (i.e., as a type II protein, with the N-terminus projecting toward the cytosol and the C-terminus projecting away from the cell surface) as compared with the orientation of Fc in the naturally type I cell surface IgG polypeptide (with the C-terminus projecting toward the cytosol and the N-terminus projecting away from the cell surface).
  • the reverse orientation results from the fusion ofthe Fc region with a type II membrane protein transmembrane domain.
  • Cell surface Fc expressed in the reverse orientation a novel design in this invention, retains the biological function of IgG, Fc of binding Fc receptor to mediate macrophage activation, while simultaneously losing the complement fixation capability, as described in EXAMPLE 1.
  • CH e.g., SEQ ID NO:2
  • CH e.g., SEQ ID NO:4
  • hinge regions e.g., SEQ ID NO:4
  • Full length IgG, heavy chain constant region (hTR-Fc) resides primarily in the endoplasmic reticulum. Removal ofthe CH, domain allows translocation the chimera from the endoplasmic reticulum to the plasma membrane.
  • Immunostimulatory cell surface polypeptides and their receptors are important for the clearance and destruction of foreign materials, including mammalian cells or bacteria. Immunostimulatory cell surface polypeptides and their receptors activate the phagocytosis and ADCC. The process begins with opsonization of the foreign materials.
  • An opsonin is an agent, usually an antibody or complement components, that makes a cell or microbe more vulnerable to being engulfed by a phagocyte; opsonization is the process of coating a cell with opsonin.
  • a phagocyte is an cell that engulfs and devours another; the process of engulfing and devouring is phagocytosis.
  • Monocytes are a type of large white blood cell that travels in the blood but which can leave the bloodstream and enter tissue to differentiate into macrophages. Macrophages digest debris and foreign cells. Monocytes are generally characterized by the cell surface expression of CD 14.
  • cells coated with immunoglobulins bind to phagocytes through the Fc receptors on the phagocytes. Phagocytes respond to signals from the Fc receptors by assembling cytoskeletal proteins, signaling cytoskeletal-protein assembly by activation of protein tyrosine kinases, and by phagocytosing the cell coated with immunoglobulin.
  • IgG-Fc ⁇ RI interaction activates various biological functions such as phagocytosis, endocytosis, ADCC, release of inflammatory mediators and superoxide anion production.
  • Macrophages possess organic anion transporter proteins that promote the afflux of anionic substances from the macrophage.
  • Fc ⁇ RI mediates ADCC by macrophages and triggers both phagocytosis and superoxide production.
  • the cells and methods of the invention where the Fc domain of IgG is expressed on the surface ofthe cell to interact with phagocyte Fc receptor cause phagocytes to bind to the cell expressing the Fc domain of IgG, inducing ADCC.
  • the IgG, and IgG 3 isotypes, that interact with the high affinity receptor Fc ⁇ RI on macrophages, are preferred for the cells and methods of the invention.
  • Macrophages can also present antigens to T cells.
  • macrophages are involved in other components ofthe immune response, including the humoral immune response (antibody production) and cellular immune response.
  • a major function associated with human IgG is the activation of complement, an undesirable characteristic for encapsulated cell therapy. Activation of complement pathways may lead to a variety of undesirable biologic actions, such as damage cells within a device.
  • the transformed cells ofthe invention have the useful characteristics of being (1) cells expressing immunostimulatory cell surface polypeptide; (2) so that the immunostimulatory cell surface polypeptide activates macrophages; but (3) the immunostimulatory cell surface polypeptide does not activate the complement cascade.
  • a C3a enzyme immunoassay showed that no complement fixation occurs (FIGURE 5).
  • Immunostimulatory cell surface polypeptide fused to a second cell surface polypeptide In one embodiment, the immunostimulatory cell surface polypeptide is fused to a second cell surface polypeptide to form a single polypeptide expressed at the cell surface.
  • the second cell surface protein anchors the immunostimulatory cell surface polypeptide to the exterior of the cell.
  • second cell surface proteins that may be suitable for such use include transferrin, CD10, CD 13, CD23, CD26, CD38, CD71, CD72, CD74, 4F2, BP-1, endoglin, Ly-49, M-ASGP-BP, NKG2A, NKR-PI, and PC-1.
  • CD71, CD72, BP- 1 , endoglin, Ly-49, NKR-PI and PC-1 are preferred because these polypeptides are known to dimerize and can facilitate dimerization of the Fc region of the antibody, for the enhanced stability of Fc.
  • the second cell surface polypeptide can be human transferrin receptor (hTR), a type II cell surface protein.
  • hTR human transferrin receptor
  • the extracellular region of the transferrin receptor substitutes for the native hinge region of IgG to anchor Fc (residues 89-97).
  • the hTR fragment is approximately equal in length, but not in amino acid identity, to native IgG, hinge, and may effectively provide spacer functions similar to that of the hinge region.
  • the hinge region of IgG provides intermolecular disulfide bonds between heavy and light chains using critical cysteine residues.
  • the hTR region (1-97) contains at least one cysteine (C89) to mimic the hinge region function, by allowing multimeric association of the hTR-Fc ⁇ H monomers .
  • transformed cells and recombinant genetic techniques A "transformed" cell is a cell or progeny of a cell into which has been introduced, by means of recombinant genetic techniques, a polynucleotide encoding a cell surface protein.
  • the term “recombinant” refers to a product of human intervention.
  • the transformed cell may be any human cell that can express an immunostimulatory cell surface polypeptide. Any suitable source of human tissue, can be used as a source for generating transformed cells, including publicly available immortalized cell lines and dividing primary cell cultures.
  • human cell lines include human neural stem or progenitor cells; RPMI 2650, HT-1080 or SW-13 epithelial cells; HL-60 macrophage cells; CCRF-CEM or RPMI 8226 lymphoid cells; and WI-38, HEL1, MRC-5 or IMR-90 fibroblast cells.
  • Useful human cell lines have the ability to be easily transfected, and to secrete proteins and peptides.
  • the transformed cells can be from other mammalian sources, for example, from rodents.
  • EXAMPLE 1 baby hamster kidney (BHK) cells opsonized with different concentrations of antibody stimulated a dose-dependent increase in superoxide production (FIGURE 3 A).
  • BHK-Fc cells induce superoxide production.
  • the presence of IgG Fc on the cell surface of transformed hamster cells activate macrophages.
  • a recombinant polynucleotide encoding an immunostimulatory cell surface polynucleotide can be constructed in a standard DNA expression vector and introduced to a cell for expression within the cell.
  • Polynucleotides for insertion into cloning vectors for example coding polynucleotides, can be constructed using the polymerase chain reaction (PCR) to amplify appropriate polynucleotides.
  • PCR polymerase chain reaction
  • Polynucleotide synthesis and purification techniques are described in Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989) and Current Protocols in Molecular Biology (Ausubel et al, Eds., Wiley Interscience, N.Y. 1993).
  • the PCR procedure is performed by well-known methodology. See, for example, Ausubel et al, and Bangham (In Protocols in Molecular Genetics, Humana Press, 1991).
  • PCR kits can be purchased from companies such as Stratagen
  • a "vector” is a replicon to which coding polynucleotide is attached, so as to bring about the replication or expression of the attached coding polynucleotide .
  • Vectors can be used for the transformation of cells in gene manipulation bearing a coding polynucleotide corresponding to appropriate polypeptides that, when combined with appropriate control sequences, confer specific properties on the transformed cell.
  • Recombinant vectors are constructed by cutting and joining polynucleotides from different sources using restriction enzymes and ligases. Vectors include cloning vectors and expression vectors.
  • a cloning vector is a polynucleotide, such as a plasmid, cosmid or bacteriophage, that can replicate autonomously in a host prokaryotic or eukaryotic cell.
  • Cloning vectors typically contain one or a small number of restriction endonuclease recognition sites at which polynucleotide sequences can be inserted in a determinable fashion without loss of an essential biological function of the vector, as well as a marker gene that is suitable for use in the identification and selection of cells transformed with the cloning vector. Suitable cloning vectors are described by Sambrook et al.
  • Cloning vectors can be obtained, for example, from GIBCO/BRL (Gaithersburg, MD), Clontech Laboratories, Inc. (Palo Alto, CA),
  • Cloned variants are amplified by transforming competent bacterial cells with a cloning vector and growing the bacterial host cells in the presence ofthe appropriate antibiotic (see, for example, Sambrook et al, and Ausubel et al., above). Bacterial host cells are then screened for the appropriate clones.
  • the resulting recombinant polynucleotide or relevant parts can be cloned from cloning vectors into expression vectors, which expression vectors have characteristics permitting higher levels of, or more efficient expression of, the resident polynucleotides.
  • These constructs may require a promoter that initiates transcription ofthe inserted coding polynucleotide.
  • promoter is a polynucleotide sufficient to direct transcription, including those promoter elements which are sufficient to render promoter-dependent gene expression inducible.
  • a polynucleotide encoding a biologically active cell surface protein polypeptide is operably linked to a promoter.
  • operably linked refers to a juxtaposition where the components are configured so as to perform their usual function.
  • promoter operably linked to a coding polynucleotide is capable of effecting the expression of the coding polynucleotide.
  • operably linked is meant that a coding polypeptide and a promoter are functionally connected to permit gene expression when the appropriate factors (e.g., transcriptional activator proteins) are bound to the regulatory sequence.
  • the orientation or placement of the elements ofthe vector is not strict, so long as the operable linkage requirement is fulfilled for control of and expression of the coding polynucleotide.
  • a “mammalian” promoter is a polynucleotide that directs transcription in a mammalian cell (e.g., a promoter of a mammal or a virus that infects a mammal).
  • Transcriptional regulatory sequences include a promoter region sufficient to direct the initiation of RNA synthesis. Suitable eukaryotic promoters include the promoter of the mouse metallothionein I gene (Hamer et al. , J. Molec. Appl. Genet. 1: 273, 1982); the TK promoter of
  • Herpes virus (McKnight, Cell 31: 355, 1982); the SV40 early promoter (Benoist et al , Nature 290: 304, 1981); the Rous sarcoma virus promoter (Gorman et al, Proc. Nat'l Acad. Sci. USA 79: 6777 1982); and the cytomegalovirus promoter (Foecking et al, Gene 45: 101 , 1980).
  • Many genetic constructs and methods for expressing heterologous genes in cells of mammals are known in the art and are suitable for use in the invention.
  • expression of a cell surface protein can be accomplished with conventional gene therapy methods, such as those that employ viral vectors (e.g., vectors derived from retroviruses, adenoviruses, herpes viruses, vaccinia viruses, polio viruses, Sindbis viruses, or adeno-associated viruses).
  • viral vectors e.g., vectors derived from retroviruses, adenoviruses, herpes viruses, vaccinia viruses, polio viruses, Sindbis viruses, or adeno-associated viruses.
  • the immunostimulatory cell surface polypeptide is constitutively expressed in the transformed cell.
  • Constitutive expression is achieved by the use of a vector with a constitutive promoter.
  • the vector pRc/CMV the vector pRc/CMV
  • the level of expression may depend on the immunostimulatory cell surface polypeptide used, on the vector copy number, or the vector cellular or genomic location, by does not, by contrast with inducible expression, depend on the addition of factors.
  • Constitutive expression can occur when the recombinant polynucleotide becomes part of the genome of an organism (i.e., either stably integrated or as a stable extrachromosomal element) that develops from that cell.
  • a polynucleotide may include a gene which is partly or entirely heterologous (i.e., foreign) to the transgenic organism, or may represent a gene homologous to an endogenous gene of the organism.
  • Increased constitutive or inducible expression can be achieved by increasing or amplifying the vector copy number using amplification methods well known in the art.
  • amplification methods include, e.g., DHFR amplification (see, e.g., Kaufman et al, United States Patent No. 4,470,461) or glutamine synthetase ("GS") amplification (see, e.g., United States Patent No. 5,122,464, and European Patent Application Publication No. EP 338,841).
  • G418 The G418 resistance gene codes for aminoglycoside phosphotransferase (APH) which enzymatically inactivates G418 added to the culture medium. Only those cells expressing the APH gene will survive drug selection usually resulting in the expression ofthe second biologic gene as well.
  • APH aminoglycoside phosphotransferase
  • the hygromycin B phosphotransferase (HBH) gene codes for an enzyme that specifically modifies hygromycin toxin and inactivates it. Genes co-transfected with or contained on the same plasmid as the hygromycin B phosphotransferase gene will be preferentially expressed in the presence of hygromycin B.
  • the expression vector encoding the immunostimulatory cell surface polypeptide is inducible. High levels of expression can be accomplished by the addition of a regulatory region which provides increased transcription of downstream sequences in the appropriate host cell.
  • the transcriptional and translational regulatory signals preferably are derived from viral sources, such as adenovirus, bovine papilloma virus, simian virus, or the like, in which the regulatory signals are associated with a particular gene which has a high level of expression.
  • viral sources such as adenovirus, bovine papilloma virus, simian virus, or the like, in which the regulatory signals are associated with a particular gene which has a high level of expression.
  • Suitable transcriptional and translational regulatory sequences also can be obtained from mammalian genes, such as actin, collagen, myosin, and metallothionein genes.
  • the invention therefore provides a "suicide gene" for use in therapy.
  • transformed cells are used to provide a pharmacologically effective treatment.
  • expression of the immunostimulatory cell surface polypeptide is induced.
  • the expression results in the transformed cell being effectively removed from the patient.
  • Expression in the central nervous system The brain is an immunologically privileged site, sheltered from circulating cells and proteins ofthe immune system; but a growing body of evidence implicates complement in numerous brain diseases (see review by Morgan et al, Immunopharmacology 38(1 -2) .43-50, 1997).
  • Complement synthesis and activation in the brain are important in immune defense at this site, but may also be of importance in CNS conditions such as Alzheimer's disease, ischaemia and Parkinson's disease, as well as in peripheral disorders such as myocardial ischaemia and xenotransplantation (McGeer & McGeer, Drugs55 (6) .139-46, 1998).
  • AD Alzheimer disease
  • C2, C3, C4, C5 C6, C7, C8 and C9
  • Some brain cells synthesize complement and also express specific receptors; some are extraordinarly sensitive to the lytic effects of complement.
  • Complement activation causes neuronal cell death in vitro, and this neurodegenerative process is regulated by homologous restriction, as described by Shen et al. (Brain Res Brain Res Protoc 1 (2) .T 86-94, 1997).
  • the cells, compositions, and methods ofthe invention are useful in modulation of the central nervous system immune response.
  • the promoter can be cell-specific, tissue-specific, or stage-specific, to express the immunostimulatory cell surface polypeptide in neural cells with increased specificity.
  • expression vectors examples include the commercially available pRC/CMV, pRC/RSV, and pCDNAlNEO (Invitrogen), where the viral promoter regions of interest are replaced with promoter sequences that are not subject to the down regulation experienced by viral promoters within the central nervous system.
  • the GFAP promoter can be employed for the transfection of astrocytes and the MBP promoter can be used in oligodendrocytes.
  • promoters include, but are not limited to, the promoters of hDBH (human dopamine beta hydroxylase) (Mercer et al, Neuron 7: 703-716, 1991)), hTH (human tyrosine hydroxylase) (Kaneda et al, Neuron 6: 583-594, 1991), hPNMT (human phenylethanolamine N-methyltransferase) (Baetge et al, PNAS, 85: 3648-3652, 1988), mGFAP (mouse glial fibrillary acidic protein) (Besnard et al, J. Biol.
  • MBP myelin basic protein
  • mNF-L mouse neurofilament-light subunit
  • hPo human P 0 , the promoter for the gene encoding the major myelin glycoprotein in the peripheral nervous system
  • mMT rat neuron-specific enolase
  • the transformed cells or immunostimulatory cell surface polypeptides ofthe invention are diagnostically useful for the detection of macrophage response to immunostimulatory cell surface polypeptides, for example, in inflammation.
  • Biological samples e.g. blood or derivatives thereof, biopsies, synovial fluid, etc.
  • Assays may be performed on cell lysates, intact cells, frozen sections, etc.
  • inflammation e.g. arthritis, bacterial infections, hypersensitivity, wound healing, etc.
  • in vitro phagocytes such as macrophages
  • in vitro cells expressing immunostimulatory cell surface polypeptides the activation of in vitro phagocytes, such as macrophages, by in vitro cells expressing immunostimulatory cell surface polypeptides is measured, as an in vitro test of a patient's macrophage ability to ingest and kill specific target cells.
  • purified or semi-purified immunostimulatory cell surface polypeptide may be bound to an insoluble substrate, and used in lieu ofthe cells or tissue.
  • the immunostimulatory cell surface polypeptides are also diagnostically useful in screening assays to determine whether an agent is effective in interfering with the interaction between phagocytes and immunostimulatory cell surface polypeptides.
  • in vitro phagocytes such as macrophages
  • in vitro cells expressing immunostimulatory cell surface polypeptides are measured.
  • Agents, particularly peptides, aptamers, carbohydrates, small organic molecules, etc. are added to the mixture of antibody and cells, and a measured reduction in phagocyte activity indicates that the compound reacts with the immunostimulatory cell surface polypeptide.
  • agent describes any molecule, e.g. protein or pharmaceutical, with the capability of altering or mimicking the physiological function of a phagocyte.
  • agent describes any molecule, e.g. protein or pharmaceutical, with the capability of altering or mimicking the physiological function of a phagocyte.
  • agent concentrations e.g. protein or pharmaceutical
  • a plurality of assay mixtures are run in parallel with different agent concentrations to obtain a differential response to the various concentrations.
  • one of these concentrations serves as a negative control, i.e. at zero concentration or below the level of detection.
  • combinatorial chemistry methods for identifying chemical compounds. See, for example, Plunkett & Ellman,
  • Candidate agents encompass numerous chemical classes, though typically they are organic molecules, preferably small organic compounds having a molecular weight of more than 50 and less than about 2,500 daltons (Da).
  • Candidate agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two ofthe functional chemical groups.
  • the candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups.
  • Candidate agents are also found among biomolecules including, but not limited to: peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof.
  • Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification or amidification to produce structural analogs.
  • the screening assay is a binding assay
  • the label can directly or indirectly provide a detectable signal.
  • Various labels include radioisotopes, fluorescers, chemiluminescers, enzymes, specific binding molecules, particles, e.g. magnetic particles, and the like.
  • Specific binding molecules include pairs, such as biotin and streptavidin, digoxin and antidigoxin.
  • the complementary member would normally be labeled with a molecule that provides for detection, in accordance with known procedures.
  • reagents may be included in the screening assay. These include reagents like salts, neutral proteins, e.g. albumin, detergents, etc that are used to facilitate optimal protein-protein binding and/or reduce non-specific or background interactions. Reagents that improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors or anti-microbial agents may be used.
  • the mixture of components are added in any order that provides for the requisite binding. Incubations are performed at any suitable temperature, typically between 4 and 40°C. Incubation periods are selected for optimum activity, but may also be optimized to facilitate rapid high-throughput screening. Typically between 0.1 and 1 hours will be sufficient.
  • Second antigen presentation The invention provides a method for antigen presentation.
  • second antigens Mildly or minimally antigenic substances
  • the second antigens are on the surface of a transformed cell containing a recombinant polynucleotide with a promoter operably linked to a polynucleotide coding for an immunostimulatory cell surface polypeptide.
  • the transformed cell containing a recombinant polynucleotide with a promoter operably linked to a polynucleotide coding for an immunostimulatory cell surface polypeptide actually makes the second antigenic substances.
  • the second antigen could be encoded by a recombinant polynucleotide that has been used to transform the cell.
  • transformed cells can be constructed by recombinant techniques to express the second antigen against which a strong immune response is desired.
  • This method of the invention is useful for promoting a significant immune response against otherwise weak antigens.
  • the weak antigens are tumor associated antigens or certain viral antigens, e.g., specific antigens of HIV- 1 , etc.
  • the invention provides a method for stimulating phagocyte activity.
  • a transformed cell containing a recombinant polynucleotide with a promoter operably linked to a polynucleotide coding for an immunostimulatory cell surface polypeptide is made to contact a phagocyte, such as a macrophage.
  • the contact stimulates an increased phagocytic activity by the phagocyte.
  • the phagocyte may engulf the transformed cell.
  • the phagocyte may also or alternatively engulf other cells, or exhibit measurable properties of activated macrophages, such as those described in EXAMPLE 1.
  • a therapeutic compound for example an anti-tumor compound, is introduced into a transformed cell containing a recombinant polynucleotide with a promoter operably linked to a polynucleotide coding for an immunostimulatory cell surface polypeptide.
  • This transformed cell is introduced into a host, such as a patient.
  • the transformed cell is targeted to the macrophagic tumor, which phagocytoses the transformed cell.
  • the therapeutic compound is delivered.
  • Modulating an immune response The invention also provides a method for modulating an immune response using transformed cells expressing immunostimulatory cell surface polypeptides.
  • modulate means that the phagocyte activity is controlled or regulated in vivo by the methods ofthe present invention.
  • modulate can mean either stimulating or inhibiting the response, depending on the situation.
  • the method ofthe invention includes treatment of conditions in which either the immune reactions are deleterious and suppression of such responses or immune reactions is desirable, or conditions in which immune reactions are important and stimulation of such responses is desirable.
  • An immunostimulatory cell surface polypeptide may be useful in recruiting or activating macrophages that would enhance the immune response to a vaccine, stimulate a response for tumor rejection, or alter the response in a qualitative manner.
  • the immunostimulatory cell surface polypeptide may inhibit or depress an immune or inflammatory response where desirable, such as in graft rejection responses after organ and tissue transplantations, or autoimmune disease.
  • organs and tissues such as kidneys, hearts, livers, skin, pancreatic islets and bone marrow.
  • Autoimmune disorders include the group of diseases caused by reactions ofthe immune system to self antigens leading to tissue destruction.
  • the immune system's response may be primarily humoral (autoantibody production), primarily cellular
  • autoreactive T-cells delay ed-type hypersensitivity T-cells and perhaps cytotoxic T-cells, i.e., "autoreactive T cells"
  • humoral and cellular reactions may be induced.
  • the highly specific reactivity of autoreactive T-cells is directed against external cell-surface structures, internal cytoplasmic or nuclear constituents, or against secreted products produced by cells in different organs.
  • self-antigen reactive T H -cells There is clearly a problem of some kind regarding the development of self-antigen reactive T H -cells.
  • expression of an immunostimulatory cell surface polypeptide results on the elimination of autoreactive t-cells, thus reducing a factor involved in the autoimmune disorder.
  • autoimmune diseases include diabetes; autoimmune thyroiditis; multiple sclerosis and related demyelinating diseases; rheumatoid arthritis; systemic lupus erythematosis; and myasthenia gravis.
  • Other autoimmune and related disorders include, e.g., polyarteritis nodosa: polymyositis and dermatomyositis: progressive systemic sclerosis (diffuse scleroderma): glomerulonephritis: Sjogren's syndrome: Hashimoto's disease and Graves' disease: adrenalitis: hypoparathyroidism: pernicious anemia; uveitis pemphigus and pemphigoid; cirrhosis and other diseases of the liver; ulcerative coliris; myocarditis; regional enteritis; adult respiratory distress syndrome; local manifestations of drug reactions (dermatitis, etc.); inflammation-associated or allergic reaction patterns of the skin; atopic dermatitis and infantile ec
  • the invention provides a composition in which transformed cells containing polynucleotides encoding an immunostimulatory cell surface polypeptide is encapsulated in an immunoisolatory capsule.
  • An "immunoisolatory capsule” means that the capsule upon implantation into a host minimizes the deleterious effects of the host's immune system on the cells within the core. In the rare event that encapsulated cells should escape from a capsule whose integrity has been breached, the cells can be immediately eliminated by the host without triggering specific immunological memory. When encapsulated, the transformed cell does not activate macrophages, but unencapsulated cells are effectively eliminated by the host. Encapsulated cell therapy is a valuable therapeutic method.
  • Encapsulated cell therapy is based on the concept of isolating cells from a host's immune system by surrounding the cells with a semipermeable biocompatible material before implantation within the host. Using encapsulation techniques, cells can be transplanted into a host without immune rejection or use of immunosuppressive drugs.
  • Useful biocompatible polymer capsules usually contain (a) a core which contains a cell or cells, either suspended in a liquid medium or immobilized within an immobilizing matrix, and (b) a surrounding or peripheral region of permselective matrix or membrane ("jacket") which does not contain isolated cells, which is biocompatible, and which is sufficient to protect isolated cells if present in the core from detrimental immunological attack.
  • Encapsulation hinders elements of the immune system from entering the capsule, thereby protecting the encapsulated cells from immune destruction.
  • the semipermeable nature ofthe capsule membrane also permits the biologically active molecule of interest to easily diffuse from the capsule into the surrounding host tissue. This technique prevents the inherent risk of tumor formation and allows the use of the transformed cells without immunosuppression of the recipient.
  • the implant may be retrieved if necessary or desired.
  • the capsule is made from a biocompatible material.
  • a "biocompatible material” is a material that, after implantation in a host, does not elicit a detrimental host response sufficient to result in the rejection of the capsule or to render it inoperable, for example through degradation.
  • the biocompatible material is relatively impermeable to large molecules, such as components of the host's immune system, but is permeable to small molecules, such as insulin, growth factors, nutrients, while metabolic waste to be removed.
  • a variety of biocompatible materials are suitable for delivery of growth factors by the composition ofthe invention. Numerous biocompatible materials are known, having various outer surface morphologies and other mechanical and structural characteristics.
  • the capsule of this invention will be similar to those described by Aebischer et al. (International Patent Application No. WO 92/19195); Baetge (International Patent Application Publication No. WO 95/05452); or United States Patent Nos.
  • Components ofthe biocompatible material may include a surrounding semipermeable membrane and the internal cell-supporting scaffolding. preferably, the transformed cells are seeded onto the scaffolding, which is encapsulated by the permselective membrane.
  • the filamentous cell-supporting scaffold may be made from any biocompatible material selected from the group consisting of acrylic, polyester, polyethylene, polypropylene polyacetonitrile, polyethylene teraphthalate, nylon, polyamides, polyurethanes, polybutester, silk, cotton, chitin, carbon, or biocompatible metals.
  • bonded fiber structures can be used for cell implantation (U.S. Patent No. 5,512,600).
  • biodegradable polymers can be use as scaffolds for hepatocytes and pancreatic cells, as reviewed by Cima et al. (Biotech. Bioeng. 38: 145-58, 1991).
  • Biodegradable polymers include those comprised of poly(lactic acid) PLA, poly(lactic-coglycolic acid) PLGA, and poly(glycolic acid) PGA and their equivalents. Foam scaffolds have been used to provide surfaces onto which transplanted cells may adhere.
  • Woven mesh tubes have been used as vascular grafts. Additionally, the core can be composed of an immobilizing matrix formed from a hydrogel, which stabilizes the position of the cells.
  • a hydrogel is a three dimensional network of cross-linked hydrophilic polymers in the form of a gel, substantially composed of water.
  • the surrounding semipermeable membrane can be used to manufacture the surrounding semipermeable membrane, including polyacrylates (including acrylic copolymers), polyvinylidenes, polyvinyl chloride copolymers, polyurethanes, polystyrenes, polyamides, cellulose acetates, cellulose nitrates, polysulfones (including polyether sulfones), polyphosphazenes, polyacrylonitriles, poly(acrylonitrile/covinyl chloride), as well as derivatives, copolymers and mixtures thereof.
  • the surrounding semipermeable membrane is a biocompatible semipermeable hollow fiber membrane.
  • the surrounding semipermeable membrane is formed from a polyether sulfone hollow fiber, such as those described by Wechs (United States Patent No. 4,976,859) and Muller & Wechs (United States Patent No. 4,968,733).
  • An alternate surrounding semipermeable membrane material is poly(acrylonitrile/covinly chloride).
  • the capsule can be any configuration appropriate for maintaining biological activity and providing access for delivery ofthe product or function, including for example, cylindrical, rectangular, disk- shaped, patch-shaped, ovoid, stellate, or spherical. Moreover, the capsule can be coiled or wrapped into a mesh-like or nested structure. If the capsule is to be retrieved after it is implanted, configurations which tend to lead to migration of the capsules from the site of implantation, such as spherical capsules small enough to travel in the recipient's blood vessels, are not preferred. Certain shapes, such as rectangles, patches, disks, cylinders, and flat sheets offer greater structural integrity and are preferable where retrieval is desired.
  • the scaffolding may be coated with extracellular matrix (ECM) molecules.
  • ECM extracellular matrix
  • Suitable examples of ECM molecules include, for example, collagen, laminin, and fibronectin.
  • the surface ofthe scaffolding may also be modified by treating with plasma irradiation to impart charge to enhance adhesion of cells.
  • any suitable method of sealing the capsules may be used, including the employment of polymer adhesives and/or crimping, knotting and heat sealing.
  • any suitable "dry” sealing method can also be used, as described, e.g., in United States Patent No. 5,653,687.
  • implantation sites are contemplated for the devices and methods of this invention. These implantation sites include the central nervous system, including the brain, spinal cord, and aqueous and vitreous humors of the eye.
  • IgG IgG
  • Fc chimera with the Fc in a reversed orientation, were constructed and successfully expressed on a cell surface.
  • the Fc receptor binding property was retained in the molecules, while the complement activation capability was absent.
  • Such a reversed Fc must be anchored by the N-terminus with the carboxyl terminus projecting away from the cell surface (FIGURE IC), in contrast to the naturally occurring membrane bound IgG, which is anchored to the membrane via the Fc portion (FIGURE IB).
  • a type II transmembrane domain is necessary to achieve such a configuration.
  • the hTR is a type II transmembrane protein with the transmembrane region also functioning as a signal peptide.
  • the transmembrane domain of the human transferrin receptor was fused in-frame to the N-terminus of the second and third domains of human immunoglobulin Gl heavy chain constant region.
  • This fusion molecule was designed to take advantage ofthe type II membrane anchor property ofthe transferrin receptor to express the Fc portion of the molecule in a reverse orientation, such that the Fc portion projected away from the cell surface. This is in contrast to the conventional cell surface IgG, which is anchored by a C-terminal type I transmembrane domain.
  • a chimeric protein containing the hTR transmembrane domain and human IgG Fc domain was designed.
  • the cell surface expressed reverse Fc no longer activated complement but retained Fc receptor binding capability, and activated superoxide production by macrophages. This activity was completely blocked by a Fc ⁇ RI-specific monoclonal antibody.
  • Oligonucleotide #402 (SEQ ID NO: 7) is specific for the human transferrin receptor and contains the synthetic BamHI restriction site, as single underlined below: #402 : 5 ' - CCC GGA TCC GCC ACC ATG ATG GAT CAA GCT AG- 3 ' Oligonucleotide #403 (SEQ ID NO: 8) is specific for the human transferrin receptor (GAC GA TGG GCC CTT GGT GGA GGC) and the human IgG, (CTC AGT TTT TGG).
  • Oligonucleotides #407 (SEQ ID NO:12) and #408 (SEQ ID NO: 13) are complementary to each other:
  • Oligonucleotide #408 has the 5' 12 nucleotides identical to the hTR sequence and the 3'
  • Oligonucleotide #407 (SEQ ID NO: 12) has the 5' 12 nucleotides identical to the mlgG, sequence and the 3' 18 nucleotides identical to the hTR sequence.
  • oligonucleotide #409 (SEQ ID NO: 14) contains an internal BamHI restriction site.
  • CH or the hinge domains
  • PCR polymerase chain reaction
  • hTR human transferrin receptor
  • Residues 1-97 of hTR, which includes the putative signal sequence and transmembrane domain, and human IgG, N-terminal deletion fragments were amplified from human spleen cDNA (Clontech, San Diego, CA) by PCR using the Advantage GC Genomic PCR kit (Clontech, San Diego, CA).
  • the IgG, region encoding the CH,, hinge, CH 2 , and CH 3 domains was generated by RT-PCR of the human and mouse spleen total RNA.
  • the source ofthe human transferrin receptor (“hTR") was the plasmid HBMAC38 (ATCC 100808) from the American Type Culture Collection ("ATCC”) in Rockville, Maryland. Briefly, 0.5 ⁇ g of total RNA was reverse transcribed to generate cDNA in a 20 ml reaction mixture according to Krug & Berger (Methods Enzymol, 152: 316-325, 1987).
  • PCR reactions were also carried out using oligonucleotide pairs #406 (SEQ ID NO:l 1) and #407 (SEQ ID NO:12); and oligonucleotide pairs #408 (SEQ ID NO: 13) and #409 (SEQ ID NO: 14); on templates HBMAC38 and pcDNA3.1(-)-mIgG, plasmids, respectively.
  • Reaction mixtures were subjected to 30 cycles of PCR. Each cycle consisted of denaturation at 94°C for 1 minute, annealing at 50°C for 1 minute, and extension at 72°C for 1 minute.
  • the PCR fragment was purified away from the used deoxynucleotides and salt in PCR reaction mixtures by the QIAquick solution purification kit (Qiagen, Chatsworth, CA) according to the manufacturer's protocol.
  • the purified human and mouse IgG, RT-PCR fragments were digested by BamHI/Hindlll and EcoRI/Hindlll, respectively, and subsequently ligated into pcDNA3.1(-) /BamHI/Hindlll and pcDNA3.1(-)/EcoRI/HindIII, respectively, which was dephosphorylated by alkaline phosphatase treatment.
  • the hTR and IgG heavy chain fragments were ligated, generating the coding sequence for the chimeric molecule. These chimeras were designated hTR-
  • Fc (containing IgG, heavy chain constant region, full length), hTR-Fc ⁇ CH, (CH, domain deleted IgG, heavy chain constant region; SEQ ID NO:l) and hTR-Fc ⁇ H (CH, and hinge deleted IgG, heavy chain constant region; SEQ ID NO:3) respectively.
  • the ligation mixtures were transformed into DH5a, and ampicillin-resistant colonies were screened for positive clones.
  • a cracking gel procedure (Promega Protocols and Applications Guide, 19991) was used to screen out the positive clones. The identity ofthe correct clones was further verified by BamHI/Dralll double digestion.
  • the plasmid DNA obtained from the ampicillin-resistant colonies were digested by BamHI/Hindlll and EcoRI/Hindlll restriction endonucleases to verify the presence of human and mouse IgG, RT-PCR fragments, respectively.
  • the resulting pcDNA3.1(-)-based human and mouse IgG, intermediate cloning vectors were named pcDNA3.1(-)-hIgG, and pcDNA3.1(-)-mIgG, respectively. Characterization and localization of the fusion proteins Transfected cells express fairly high levels of intact hTR-Fc ⁇ H(SEQ ID NO:4).
  • BHK cells also expressed hTR-Fc and hTR-Fc ⁇ CH, (SEQ ID NO:2) that were shown to have approximately the expected molecular mass.
  • the transfection of baby hamster kidney (BHK) cells was performed by plating on 6-well tissue culture plates (Fisher Scientific, Pittsburgh, PA) or Lab-Tek chamber slides (Nunc, Napierville, IL) coated with poly-omithine (Sigma, St. Louis, MO). Cells were transfected using the calcium phosphate-based Stable Transfection Kit (Stratagene, San Diego, CA), using 1.0 mg DNA per ml of media per well.
  • DMEM Dulbecco's Modified Essential Medium
  • FBS heat inactivated fetal bovine serum
  • hTR-Fc ⁇ H SEQ ID NO:4
  • surface expression of reverse human IgG Fc was monitored by both immunostaining and ELISA.
  • immunofluorescence staining cells were processed essentially as described by Richards et. al. (J Cell Biol, 134: 1157-68, 1996). Chimeric hTR-Fc molecules were visualized using goat anti-human IgG Fc specific C ⁇ 3 (carbocyanine) linked antibody (Jackson ImmunoResearch, West Grove, PA) diluted at 1 : 1000. Nuclei were visualized by DAPI staining.
  • a stable BHK cell line expressing hTR-Fc ⁇ H (SEQ ID NO:4) produced high levels of the reversed Fc, while wild-type cells exhibited no observable signal. Similar membrane targeting was seen with the CH, -deletion mutant (hTR-Fc ⁇ CH,; SEQ ID NO:2), whereas intact IgG Fc with only Fab deletion (hTR-Fc) targeted to the endoplasmic reticulum almost exclusively.
  • Surface expression of reverse Fc was semi-quantitated by ELISA. For ELISA, cells were seeded at 100,000/well in a 96-well tissue culture plate, as described by Margulies (In: Current Protocols in Immunology, Vol.
  • mouse macrophages were elicited by thioglycollate broth in nude mice (N:NIH(s)-nu/nuDF (Taconic Farms, Germantown, NY).
  • Mouse peritoneal macrophages were collected 72 hours after thioglycollate broth injection and resuspended into 5 x 10 6 cells/ml in RPMI + 10% FCS.
  • Fifty ml per well of cells were seeded in a 96-well tissue culture plate and the plate was incubated at 37°C for at least 3 hours. The non-adherent cells were removed by washing three time with HBSS and 50 ml/well of HBSS were added.
  • BHK cells opsonized with different concentrations of antibodies produced a dose-dependent increase in superoxide production, while control cells alone had no effect.
  • hTR-Fc fusion proteins (hTR-Fc, hTR-Fc ⁇ CH, [SEQ ID NO:2] and hTR-Fc ⁇ H [SEQ ID NO:4]) were expressed transiently or stably in BHK cells.
  • the stable clones were derived from hTR-Fc ⁇ CH, [SEQ ID NO:l]and hTR-Fc ⁇ H [SEQ ID NO:3] transfected polyclonal cells, and the effect of these clones on superoxide production by mouse macrophages are shown in FIGURE 3B. All subclones induced superoxide production. hTR-Fc did not express cell surface Fc therefore was eliminated from the study.
  • BHK-Fc ⁇ H clone 3 was chosen for further characterization.
  • 25,000 cells/well of either BHK-WT (control), opsonized BHK cells, or BHK-reverse Fc clones were added to the assay plate containing either human monocyte-like U937 cells (ATCC, Rockville, MD) pre treated with recombinant human interferon- ⁇ (R&D Systems, Minneapolis, MN) or lavaged mouse peritoneal macrophages.
  • the O 2 - release was measured as the superoxide dismutase (SOD) inhibitable reduction of cytochrome c at 550 nm by using a ThermoMax Plate reader (Molecular Devices, Menlo Park, CA). The rate of O 2 - production was monitored from time 0 to 60 mm after stimulation.
  • BHK-Fc ⁇ H(3) cells induced an increase in superoxide production, while wild-type cells had no effect.
  • C3a enzyme immunoassays can be performed as described by Burger et al. (J Immunol, 141: 553-8, 1988); Mollnes et al. (Olin Exp Immunol, 73: 484-8, 1988); and Hugh (In
  • BHK wild type (BHK-WT) and BHK-Fc ⁇ H (SEQ ID NO:4) cells were used as target cells in an ADCC assay. All testing samples were set up in two groups of triplicates and assayed employing a standard National Institutes of Health (NIH) tissue typing technique (American Society for Histocompatibility and Immunogenetics (ASHI) Manual, 1994). One group of triplicates was analyzed with sheep hypersensitized serum and
  • BHK-WT cells while the second group of triplicates was assayed in the same manner with the addition of exogenously prescreened rabbit complement.
  • the assay was performed in the absence or presence of exogenous prescreened rabbit complement. Samples were prepared using a two-color immunofluorescent microcytotoxic analysis procedure. An one- hour incubation of 1 ml sheep serum and 1 ml containing 1 ,000 BHK cells was followed by a second one-hour incubation with or without additional 5 ml rabbit complement.
  • the IgG, heavy chain with CH, deletion (SEQ ID NO:2) or CH, -hinge deletion (SEQ ID NO:4) gave similar levels of macrophage activation.
  • the extracellular region of the transferrin receptor used to anchor Fc (residues 89-97) is able to substitute for native hinge.
  • the hTR fragment used in this study is approximately equal in length, if not amino acid identity, to native IgG, hinge, _ 3Q _
  • the hTR (1-97) which contains at least one cysteine (C89) necessary for TR dimerization, is able to mimic hinge by allowing multimer association ofthe hTR-Fc ⁇ H monomers .
  • Transformed cells from EXAMPLE 1 are encapsulated for transplantation in a host.
  • the encapsulated cell devices typically include:
  • the morphology ofthe device is as follows:
  • the inner surface has a permselective skin.
  • the wall has an open cell foam structure.
  • the outer surface has an open structure, with pores up to 1.5 ⁇ m occupying 30 ⁇ 5% ofthe outer surface.
  • Hollow fibers are fabricated from PES with an outside diameter of 720 mm and a wall thickness of 100 mm (AKZO-Nobel Wippertal, Germany). These fibers are described in United States Patent Nos. 4,976,859 and 4,968,733.
  • PES#5 membrane which has a MWGO of about 280 kilodalton (kDa)
  • a PES#8 membrane which has a MWGO of about 90 kDa, is at other times used.
  • the semipermeable PES membranes typically have the following characteristics:
  • the components ofthe device are commercially available.
  • the LCM glue is available from Ablestik Laboratories (Newark, DE); Luxtrak Adhesives LCM23 and LGM24).
  • the tether material is available from Specialty Silicone Fabricators (Robles, GA). The tether dimensions are
  • the encapsulation procedure is as follows: Fiber material is first cut into 5 cm long segments and the distal extremity of each segment sealed with a photopolymerized acrylic glue (LCM-25, ICI). Following sterilization with ethylene oxide and outgassing, the fiber segments are loaded with a suspension of about 2 x 10 5 transfected cells in a collagen solution (Zyderm® soluble bovine collagen) by a Hamilton syringe and a 25 gauge needle through an attached injection port. The proximal end ofthe capsule was sealed with the same acrylic glue. Sometimes, the collagen matrix was ZyplastTM. The volume ofthe device for human use is approximately 15-18 ⁇ l. A silicone tether (Specialty Silicone Fabrication, Taunton, MA) (ID: 690 ⁇ m; OD: 1.25 mm) is placed over the proximal end of the fiber allowing easy manipulation and retrieval ofthe device.
  • a silicone tether (Specialty Silicone Fabrication, Taunton, MA) (ID: 690 ⁇ m

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Medicinal Chemistry (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Cell Biology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Toxicology (AREA)
  • Microbiology (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Physics & Mathematics (AREA)
  • Food Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Diabetes (AREA)
  • Dermatology (AREA)
  • Orthopedic Medicine & Surgery (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Neurosurgery (AREA)
  • Neurology (AREA)
EP99971035A 1998-10-26 1999-10-21 Durch zelloberflächenmoleküle induzierte makrophagenaktivierung Expired - Lifetime EP1124959B1 (de)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US178869 1998-10-26
US09/178,869 US6197294B1 (en) 1998-10-26 1998-10-26 Cell surface molecule-induced macrophage activation
PCT/US1999/024630 WO2000024897A1 (en) 1998-10-26 1999-10-21 Cell surface molecule-induced macrophage activation

Publications (2)

Publication Number Publication Date
EP1124959A1 true EP1124959A1 (de) 2001-08-22
EP1124959B1 EP1124959B1 (de) 2007-02-14

Family

ID=22654240

Family Applications (1)

Application Number Title Priority Date Filing Date
EP99971035A Expired - Lifetime EP1124959B1 (de) 1998-10-26 1999-10-21 Durch zelloberflächenmoleküle induzierte makrophagenaktivierung

Country Status (8)

Country Link
US (4) US6197294B1 (de)
EP (1) EP1124959B1 (de)
JP (1) JP4456275B2 (de)
AT (1) ATE353955T1 (de)
AU (1) AU775417B2 (de)
CA (1) CA2347928C (de)
DE (1) DE69935152T2 (de)
WO (1) WO2000024897A1 (de)

Families Citing this family (38)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6197294B1 (en) * 1998-10-26 2001-03-06 Neurotech S.A. Cell surface molecule-induced macrophage activation
US6878543B1 (en) 1999-10-25 2005-04-12 Nsgene Sa Cultures of GFAP+ nestin+ cells that differentiate to neurons
US7332168B2 (en) * 2000-08-22 2008-02-19 Micromet Ag Composition for the elimination of autoreactive B-cells
US7829084B2 (en) * 2001-01-17 2010-11-09 Trubion Pharmaceuticals, Inc. Binding constructs and methods for use thereof
US7754208B2 (en) 2001-01-17 2010-07-13 Trubion Pharmaceuticals, Inc. Binding domain-immunoglobulin fusion proteins
KR100453877B1 (ko) 2001-07-26 2004-10-20 메덱스젠 주식회사 연쇄체화에 의한 면역 글로블린 융합 단백질의 제조 방법 및 이 방법에 의해 제조된 ＴＮＦＲ/Ｆｃ 융합 단백질, 상기 단백질을 코딩하는 ＤＮＡ, 상기 ＤＮＡ를 포함하는벡터, 및 상기 벡터에 의한 형질전환체
DK1458853T3 (da) 2002-05-16 2010-04-06 Absorber Ab Fremgangsmåde til donorspecifik krydsmatchning
US20040009528A1 (en) * 2002-07-11 2004-01-15 Shyh-Yu Shaw Protein chips
EP1407765A1 (de) * 2002-10-10 2004-04-14 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Vesikel mit chimären Rezeptoren zur Induktion einer gerichteten T-Zellantwort in vivo
EP1447079A1 (de) * 2003-02-15 2004-08-18 Fraunhofer-Gesellschaft für angewandte Forschung e.V. Vesikel mit chimären Rezeptoren zur Induktion einer gerichteten T-Zellantwort in vivo
US7592143B2 (en) * 2003-04-18 2009-09-22 Cytovia, Inc. Methods of treating diseases responsive to induction of apoptosis and screening assays
EP1649000A4 (de) * 2003-06-30 2008-04-30 Lifescan Inc Aussetzen von pankreaszellen auf porösen matrizes
EP3192872A1 (de) 2003-08-26 2017-07-19 The Regents of the University of Colorado, a body corporate Inhibitoren der serinproteaseaktivität und deren verwendung in verfahren und zusammensetzungen zur behandlung bakterieller infektionen
DK1709161T3 (da) * 2004-01-19 2009-01-26 Nsgene As Humane terapeutiske celler, som udskiller nervevækstfaktor
KR100545720B1 (ko) * 2004-05-31 2006-01-24 메덱스젠 주식회사 당화된 면역글로불린 및 이를 포함하는 면역접합체
JP5188804B2 (ja) * 2004-08-03 2013-04-24 トランステック ファーマ，インコーポレイティド Ｒａｇｅ融合タンパク質及びその使用方法
UA97469C2 (uk) * 2005-07-25 2012-02-27 Емерджент Продакт Дівелопмент Сіетл, Елелсі Гуманізована специфічна до cd37 зв'язувальна молекула імуноглобуліну
EP1940471A1 (de) * 2005-10-28 2008-07-09 NsGene A/S Implantierbares biokompatibles immunisolatorisches vehikel zur abgabe von gdnf
AU2006327353B2 (en) * 2005-12-23 2011-11-24 Gcoder Systems Ab Positioning pattern
NZ569545A (en) * 2006-02-09 2011-11-25 Transtech Pharma Inc Rage fusion proteins and methods of use for treating inflammation
BRPI0711193A2 (pt) * 2006-05-05 2013-06-18 Transtech Pharma Inc proteÍnas de fusço rage, formulaÇÕes e mÉtodos de uso dos mesmos
NZ573646A (en) 2006-06-12 2012-04-27 Wyeth Llc Single-chain multivalent binding proteins with effector function
WO2008025068A1 (en) 2006-08-29 2008-03-06 Commonwealth Scientific And Industrial Research Organisation Synthesis of fatty acids
WO2008100470A2 (en) * 2007-02-15 2008-08-21 Transtech Pharma, Inc. Rage - immunoglobulin fusion proteins
WO2009023386A2 (en) * 2007-07-06 2009-02-19 Trubion Pharmaceuticals, Inc. Binding peptides having a c-terminally disposed specific binding domain
NZ603059A (en) * 2008-04-11 2014-07-25 Emergent Product Dev Seattle Cd37 immunotherapeutic and combination with bifunctional chemotherapeutic thereof
EP2389191A2 (de) 2009-01-23 2011-11-30 NsGene A/S Expression von neuropeptiden in säugetierzellen
CN102361971A (zh) 2009-01-23 2012-02-22 Ns基因公司 改善的细胞系以及在胶囊化细胞生物递送中的用途
KR20110139292A (ko) 2009-04-20 2011-12-28 화이자 인코포레이티드 단백질 글리코실화의 제어 및 그와 관련된 조성물 및 방법
CA2789446A1 (en) * 2010-02-12 2011-08-18 Oncomed Pharmaceuticals, Inc. Methods for identifying and isolating cells expressing a polypeptide
US9938353B2 (en) 2011-06-24 2018-04-10 The Regents Of The University Of Colorado, A Body Corporate Compositions, methods and uses for alpha-1 antitrypsin fusion molecules
CA2896951A1 (en) 2012-01-10 2013-07-18 The Regents Of The University Of Colorado, A Body Corporate Compositions, methods and uses for alpha-1 antitrypsin fusion molecules
WO2014022376A2 (en) 2012-08-01 2014-02-06 United Therapeutics Corporation Treatment of pulmonary arterial hypertension with prostacyclin-treated endothelial progenitor cells
JP6901823B2 (ja) 2012-08-01 2021-07-14 ユナイテッド セラピューティクス コーポレイション 間葉系幹細胞による肺動脈性高血圧症の処置
EP3878452B1 (de) 2013-01-09 2023-09-20 United Therapeutics Corporation Behandlung von vaskulopathie mit prostacyclin und mesenchymalen stammzellen
US20170007685A1 (en) * 2013-11-05 2017-01-12 The Board Of Regents Of The University Of Texas System TUMORS EXPRESSING IgG1 Fc INDUCE ROBUST CD8 T CELL RESPONSES
US11352426B2 (en) 2015-09-21 2022-06-07 Aptevo Research And Development Llc CD3 binding polypeptides
IL266175B2 (en) 2016-10-24 2024-01-01 United Therapeutics Corp Enhancement of immunomodulatory properties of MSC using treprostinil

Family Cites Families (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DK0465535T3 (da) 1989-04-03 1999-03-01 Janssen Pharmaceutica Nv Regioselektive substitutioner i cyclodextriner
CA2186528A1 (en) 1994-03-31 1995-10-12 E. Michael Egan Genetically modified cells for use in transplantation
EP0764207A1 (de) 1994-06-03 1997-03-26 Diacrin, Inc. Modifizierte zellen und verfahren zur inhibierung der hyperakuten abstossung von xenogene tranplantaten
AU2828795A (en) 1994-06-17 1996-01-15 Alexion Pharmaceuticals, Inc. Monoclonal antibodies for the reduction of anti-xenogeneic immune responses
US5795790A (en) 1994-07-20 1998-08-18 Cytotherapeutics, Inc. Method for controlling proliferation and differentiation of cells encapsulated within bioartificial organs
JPH10507906A (ja) 1994-08-19 1998-08-04 ザ ジェネラル ホスピタル コーポレイション 遺伝子工学処理したブタ細胞
WO1996006642A1 (en) 1994-08-26 1996-03-07 The General Hospital Corporation Allogeneic and xenogeneic transplantation
US6204053B1 (en) 1994-11-08 2001-03-20 Diacrin, Inc. Porcine cortical cells and their use in treatment of neurological deficits due to neurodegenerative diseases
KR19980701317A (ko) 1995-01-13 1998-05-15 돌핀 데이비드 이식 거부반응의 예방법(method to prevent transplant rejection)
CA2232815A1 (en) 1995-09-27 1997-04-03 Emory University Method of inhibiting immune system destruction of transplanted viable cells
US6166288A (en) 1995-09-27 2000-12-26 Nextran Inc. Method of producing transgenic animals for xenotransplantation expressing both an enzyme masking or reducing the level of the gal epitope and a complement inhibitor
US6197294B1 (en) * 1998-10-26 2001-03-06 Neurotech S.A. Cell surface molecule-induced macrophage activation

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO0024897A1 *

Also Published As

Publication number Publication date
DE69935152D1 (de) 2007-03-29
AU775417B2 (en) 2004-07-29
AU1216900A (en) 2000-05-15
US20010046490A1 (en) 2001-11-29
CA2347928C (en) 2012-04-17
CA2347928A1 (en) 2000-05-04
US7189837B2 (en) 2007-03-13
US6225448B1 (en) 2001-05-01
US6506891B2 (en) 2003-01-14
JP4456275B2 (ja) 2010-04-28
JP2002528078A (ja) 2002-09-03
US20030120059A1 (en) 2003-06-26
ATE353955T1 (de) 2007-03-15
WO2000024897B1 (en) 2000-07-13
WO2000024897A1 (en) 2000-05-04
US6197294B1 (en) 2001-03-06
DE69935152T2 (de) 2007-11-22
EP1124959B1 (de) 2007-02-14

Similar Documents

Publication Publication Date Title
EP1124959B1 (de) Durch zelloberflächenmoleküle induzierte makrophagenaktivierung
KR100922809B1 (ko) 면역 반응을 유발하는 항원을 동정하기 위한 조성물과 방법
JP3581366B2 (ja) 免疫原のリソソーム標的
US5525503A (en) Signal transduction via CD28
JP2008073041A (ja) 組換え型ヒトα−フェトプロテインおよびその利用
EP2248899B1 (de) NOGO Rezeptor bindendes Protein
PT1964852E (pt) Anticorpo anti-ilt7
US8182809B1 (en) Methods for treating cancer by inhibiting MIC shedding
US20100310562A1 (en) System for delivery into xcr1 positive cell and uses thereof
WO2002061105A9 (en) Chimeric molecules containing a module able to target specific cells and a module regulating the apoptogenic function of the permeability transition pore complex (ptpc)
CA2250394A1 (en) Method for the production of activated marked tumor-specific t cells and use thereof in treatment of tumors
JPH11511650A (ja) 改変されたミエリンタンパク質分子
JP2004500099A (ja) ホスファチジルセリンレセプターおよびその使用
EP1641491B1 (de) Erhöhte t-zell tumor-eindringung durch light- mutanten
US20030133909A1 (en) Nucleic acid vaccination
EP1991577A2 (de) Modulation der mdl-1-aktivität zur behandlung entzündlicher krankheiten
US20030144236A1 (en) Novel specific inhibitor of the cyclin kinase inhibitor p21 (wafl/cip1)
WO1998058541A1 (en) Modulation of apoptosis
JP2020508642A (ja) 耐性を誘導するための操作された細胞
JP2003252802A (ja) ヒト滑膜肉腫の治療薬剤

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20010523

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE

AX Request for extension of the european patent

Free format text: AL;LT;LV;MK;RO;SI

17Q First examination report despatched

Effective date: 20031218

RAP1 Party data changed (applicant data changed or rights of an application transferred)

Owner name: NEUROTECH S.A.

GRAP Despatch of communication of intention to grant a patent

Free format text: ORIGINAL CODE: EPIDOSNIGR1

RIC1 Information provided on ipc code assigned before grant

Ipc: A61K 9/48 20060101ALI20060217BHEP

Ipc: A61K 45/00 20060101ALI20060217BHEP

Ipc: G01N 33/53 20060101ALI20060217BHEP

Ipc: C12N 15/62 20060101ALI20060217BHEP

Ipc: C12N 5/10 20060101AFI20060217BHEP

GRAS Grant fee paid

Free format text: ORIGINAL CODE: EPIDOSNIGR3

GRAA (expected) grant

Free format text: ORIGINAL CODE: 0009210

AK Designated contracting states

Kind code of ref document: B1

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: NL

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20070214

Ref country code: FI

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20070214

Ref country code: DK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20070214

REG Reference to a national code

Ref country code: GB

Ref legal event code: FG4D

REG Reference to a national code

Ref country code: CH

Ref legal event code: EP

REF Corresponds to:

Ref document number: 69935152

Country of ref document: DE

Date of ref document: 20070329

Kind code of ref document: P

REG Reference to a national code

Ref country code: IE

Ref legal event code: FG4D

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: SE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20070514

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: ES

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20070525

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: PT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20070716

NLV1 Nl: lapsed or annulled due to failure to fulfill the requirements of art. 29p and 29m of the patents act
ET Fr: translation filed
PLBE No opposition filed within time limit

Free format text: ORIGINAL CODE: 0009261

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: NO OPPOSITION FILED WITHIN TIME LIMIT

26N No opposition filed

Effective date: 20071115

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: IT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20070214

Ref country code: GR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20070515

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: MC

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20071031

REG Reference to a national code

Ref country code: GB

Ref legal event code: 732E

REG Reference to a national code

Ref country code: FR

Ref legal event code: TP

REG Reference to a national code

Ref country code: CH

Ref legal event code: PUE

Owner name: NEUROTECH USA, INC.

Free format text: NEUROTECH S.A.#CHNO DES QUINZE-VINGTS RESIDENCE SAINT LOUIS 28, RUE DE CHARENTON#75571 PARIS CEDEX 12 (FR) -TRANSFER TO- NEUROTECH USA, INC.#6 BLACKSTONE VALLEY PLACE, SUITE 500#LINCOLN, RHODE ISLAND 02865 (US)

Ref country code: CH

Ref legal event code: NV

Representative=s name: BOHEST AG

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: CY

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20070214

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: LU

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20071021

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: FR

Payment date: 20121107

Year of fee payment: 14

Ref country code: CH

Payment date: 20121025

Year of fee payment: 14

Ref country code: BE

Payment date: 20121025

Year of fee payment: 14

Ref country code: IE

Payment date: 20121025

Year of fee payment: 14

Ref country code: DE

Payment date: 20121029

Year of fee payment: 14

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: GB

Payment date: 20121025

Year of fee payment: 14

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: AT

Payment date: 20121003

Year of fee payment: 14

BERE Be: lapsed

Owner name: NEUROTECH USA INC.

Effective date: 20131031

REG Reference to a national code

Ref country code: CH

Ref legal event code: PL

REG Reference to a national code

Ref country code: AT

Ref legal event code: MM01

Ref document number: 353955

Country of ref document: AT

Kind code of ref document: T

Effective date: 20131021

GBPC Gb: european patent ceased through non-payment of renewal fee

Effective date: 20131021

REG Reference to a national code

Ref country code: IE

Ref legal event code: MM4A

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: LI

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20131031

Ref country code: CH

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20131031

Ref country code: GB

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20131021

REG Reference to a national code

Ref country code: FR

Ref legal event code: ST

Effective date: 20140630

REG Reference to a national code

Ref country code: DE

Ref legal event code: R119

Ref document number: 69935152

Country of ref document: DE

Effective date: 20140501

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: AT

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20131021

Ref country code: FR

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20131031

Ref country code: DE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20140501

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: BE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20131031

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: IE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20131021